The long term objective of our research is to understand the way in which the nuclear pore controls the molecular traffic entering and exiting the nucleus. In recent years, there has been an explosion in knowledge of the events which occur in the transduction of a signal from the plasma membrane to the nucleus. Increasingly, questions of signal transduction, developmental determination, oncogenic transformation, and HIV-1 viral infectivity are focusing on nuclear import or export. Although we now have a conceptual framework for the action of the nuclear pore, we know little of the molecular nature of the pore or how it controls these myriad events. To get at that nature, in the past grant period we developed a system by which the nuclear pore itself can be reconstituted. Through immunodepletion of the system, biochemically altered nuclear pores can be created and analyzed for function. Using the system, it has been possible to identify a subunit of the nuclear pore, the p62-p58-p54 complex, and show that this subunit is required for functional nuclear pores. Our studies in this proposal focus on three broad areas: 1) A detailed analysis of this newly discovered nuclear pore subunit, the p62-58-54 complex. Specifically, we will determine the molecular structure of the complex, create mutant complex forms, and use those mutant complexes to address how the complex is assembled. From there, we will ask how the complex further assembles into the nuclear pore, and once there, what role it plays in nuclear transport. 2) A second major goal is to initiate a search for new proteins of the nuclear pore. Although the nuclear pore would be estimated to contain ~1000 total proteins, perhaps less than or equal to 60 of which are different, only 6 pore proteins have been conclusively identified. In a biochemical approach to this deficit, a newly identified Xenopus pore protein, p200, will be analyzed, as well as its companion proteins, which together comprise a second subunit of the nuclear pore. The promise of these experiments is that as each new pore protein is identified, it can immediately be tested with the nuclear pore reconstitution system for its role in pore function. A powerful genetic screen will also be carried out to identify as yet undiscovered pore proteins in yeast. 3) Lastly, the disassembly and assembly of the nuclear pore will be studied using a cell-free system which cycles between interphase and mitosis. A newly discovered phosphorylation of pore proteins which occurs at mitosis will be the focus of this study. Together, these experiments should elucidate the structure and function of the nuclear pore, the way in which it is regulated, and the mechanism of its disassembly and reformation at mitosis.